Effect of phosphatidylcholine on pld gene expression level of Aspergillus fumigatus by the real time PCR method and investigations of these genes using bioinformatics analysis.

Background and Objectives Phospholipases are a group of enzymes that breakdown phospholipid molecules producing second products. These second products play a diverse role in the cell such as signal transduction and digestion in humans. In this study, the effect of phospholipids on the expression of pld genes of A. fumigatus was investigated. The pld genes of this fungus were also investigated using bioinformatics studies. Materials and Methods Real-time PCR was performed to study the expression of pld genes. These genes were investigated using bioinformatics studies. Results There was more significant expression for all three pld genes when A. fumigatus was grown in the presence of phospholipids in the medium. The sequence of pld genes of A. fumigatus was also interrogated using bioinformatics analysis and their relationship with the other microorganisms was investigated. The fungal pld genes were more closely related to pld genes from animals and least related to bacterial pld genes. Conclusion afpld1, afpld2 and afpld3 are expressed and are up-regulated by phosphatidylcholine. Although indirect evidence of extracellular PLD activity in A. fumigatus was demonstrated, conclusive proof by partially sequencing the isolated protein will be needed and its significance in pathogenicity will have to be assessed by constructing a knockout strain and testing its virulence in a mouse model.


INTRODUCTION
One of the most prevalent opportunistic human fungal pathogens is A. fumigatus. The most serious form of invasive aspergillosis has been found in 4% of all dying patients in a modern European teaching hospital (1). A. fumigatus is a naturally thermo tolerant, saprophytic fungus which is frequently found on a wide variety of dead organic material (2). The conidia of A. fumigatus are one of the most ubiquitous in the atmosphere (3). The conidia range from 2.5 -3 µm in diameter and because of their small size they are able to enter the alveoli of the lung (2, 4). Phospholipase D (PLD) acts mainly on choline-containing phospholipids and leads to the release of choline and phosphatidic acid as hydrolysis products. In mammalian cells, two isoforms of PLD have been identified while up to three isoforms have been identified in plants (5)(6)(7). To date, most of the PLD activity described in fungi has been intracellular (8). Pld genes of this fungus regulate their internalization into the lung epithelial cells and suggest their role in pathogenicity (2).
Spore suspension was serially diluted to 10 -4 , 10 -5 and 10 -6 spores / ml -1 and plated into Petri dishes and incubated overnight at 37˚C. For liquid cultures, 50 ml of Vogel's media with or without of phospholipid, were distributed into 250 ml Erlenmeyer flasks and inoculated with 0.1 ml of a 1x 10 8 / ml -1 spore suspension and incubated with shaking (250 rpm) at 37˚C up to 24 h.
Comparative C T method, statistical and bioinformatics analysis. The comparative CT method was used to measure the level expression of the target gene by real-time PCR. CT values for gene are normalised against β-tubulin (reference housekeeping gene) to give the normalised ΔC T value.
ΔC T = C T Target -C T Reference Standard deviation for the ΔC T values were calculated using the following equation: (9) SDΔC T = (SD C T Target 2 -SD C T Reference 2 ) 1/2 To compare the relative expression of a gene grown on phosphatidylcholine compared to absence of phosphatidylcholine, the ΔC T value of the gene grown on lecithin is first subtracted from the ΔC T value of the gene grown on absence of lecithin to give the ΔΔC T value.
ΔΔC T = ΔCT absence of phosphatidylcholine -ΔC T phosphatidylcholine The fold difference in gene expression when grown on phosphatidylcholine compared to absence of phosphatidylcholine is then calculated using the formula below: (9).
Fold change in gene expression = 2 ΔΔC T As the ΔC T phosphatidylcholine value is subtracted from the ΔC T absence of phosphatidylcholine value, the standard deviation of ΔΔC T is the same as the standard deviation of ΔC T absence of phosphatidylcholine.
For comparing the statistical significance of the fold changes, the student t test was used to compare the ΔC T absence of phosphatidylcholine and ΔC T phosphatidylcholine values of each gene.
DNA sequences were submitted for analysis through the Basic Local Alignment Search Tool (BLAST). BLAST was accessed through the National Centre for Biotechnology Information (NCBI) or through the Bioinformatics Centre, University of Kyoto and used to search for sequence similarities within sequences held on the BLAST database.

RESULTS
Bioinformatics analysis of the TIGR Aspergillus fumigatus database for pld homologues. At the time this work was conducted, the unannotated genomic DNA sequence of A. fumigatus from the TIGR institute was publicly available as a series of unassembled variable lengths of contiguous DNA through the TIGR website which could be searched with basic Blast programs. While some fungal sequences and reports of pld activity were published or present in public databases, all were intracellular although there was some evidence of a secreted extracellular PLD activity in the culture supernatant from A. fumigatus (2). In addition, a partial pld gene had previously been identified from A. fumigatus (10). The aim of this work was to identify gene(s) encoding pld in the genome of A. fumigatus using bioinformatics and to make a comparison with orthologues from other published genomes (11).
Using PCR based on conserved regions of pld from other fungi, a partial homologue had previously been identified in A. fumigatus (12). The translated amino acid sequence of this homologue was used in http://ijm.tums.ac.ir a number of blast search using NCBI and the TIGR A. fumigatus genomic database. Three gene sequences were identified (Table 1).

Comparison of pld Genes from fungi and other organisms.
A phylogenetic analysis of all available pld protein sequences from bacteria, plants, animals along with derived sequences for the fungi was constructed using ClustalW version 1.7 and a phenogram created by the nearest joining method available from the European Molecular Biology Network (Fig. 1). With the exception of the two plds from A. gossypii and S. pombe, the fungal plds were more closely related to plds from animals and least related to bacterial plds.
Fungal pld's are in red, plant pld's in green, bacterial pld's in blue and animal pld's in purple. Codes represent SwissProt accession numbers. T. reesei and M. griseae were derived from genome sequence and no accession number was available.

Expression of pld genes by real-time PCR.
Expression of pld genes of A. fumigatus were measured by real time PCR using cDNA and primers which mentioned in section ( Table 2). The expression was determined using mRNA from A. fumigatus midlog phase cultures grown at 37˚C. Results are pooled from two independent experiments each with at least five replicates normalized to β-tubulin.

DISCUSSION
Real-time PCR is being increasingly employed to quantify levels of gene expression in a range of fungi (13-16). As shown in Table 2, afpld1, afpld2 and afpld3 are expressed and are up-regulated by phosphatidylcholine.
As the main site of infection of A. fumigatus is through the inhalation of spores which lodge in the lung and are therefore exposed to a phospholipid rich environment, it would appear that many of the extracellular phospholipases are likely to be upregulated following inhalation.
However, real time PCR on infected lung tissue will need to be performed to confirm if any upregulation in vitro. A. fumigatus also secretes PLD (17, 18) and these may be of great significance in lung colonisation.
PLD is known to be a major pathogenic factor in some bacterial pathogens where it causes cell lysis and tissue damage (19). In filamentous fungi, it is usually the case that genes encoding extracellular hydrolyses are normally not expressed or expressed at low levels in the absence of the substrate (20, 21).  PLD was first discovered in plants as an enzyme with phospholipid-specific phosphodiesterase activity, hydrolysing phosphatidylcholine to phosphatidic acid and choline (22). In higher eukaryotes, studies have shown that this enzyme has a rapid activation in response to extracellular stimuli and T.reesei and M.griseae were derived from genome sequence and no accession number was available.  (22). PLD is a phospholipid degrading enzyme that generates biologically active products, principally phosphatidic acid which play important functions in cell regulation. In common with many esterases, in addition to hydrolysis, PLD also catalyses a trans-esterification reaction, utilizing short-chain primary alcohols as phosphatidyl-group acceptors. (22). A number of lipid-derived molecules regulate cell proliferation and cell differentiation and specific bioactive lipids serve both as extracellular messengers on cell-surface receptors and as intracellular messengers (23). These messengers are derived from lipid constituents of biological membranes by a number of signal-activated enzymes including phospholipases, lipid kinases, and acylases. These enzymes are activated in response to receptor occupancy (24).
PLD hydrolyses phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol to form phosphatidic acid and the head group of the phospholipid substrate (25). Surveys have shown that PLD is regulated by several mechanisms in vivo (26).
Intracellular PLD activity has many cellular roles including vesicle formation, protein transport, signal transduction and mitosis (27). Phosphatidic acid, released as a result of PLD activity is itself a second messenger implicated in a variety of cellular events and signal pathway activation (27). In the fungi, PLD is required for the dimorphic transition from the yeast to the hyphal form in C. albicans and is required for mitosis in S. cerevisiae (27). In Cryptococcus neoformans, the cause of cryptococcal meningitis, there is a correlation between PLD production and the size of the capsule in strains isolated from AIDS patients (17). In the present study, the expression level of pld genes of A. fumigatus was investigated using quantifiable PCR.
In this project, a partial pld sequence which had been cloned previously by degenerate PCR (10) was used to investigate the A. fumigatus genome sequence in the TIGR database and three pld genes were identified and were translated after identifying putative introns using Gene finder software optimised for A. niger. All three translated sequences showed significant identity to yeast pld and contained two highly conserved pld active site motifs. In addition, Afpld 3 contained a phox-like domain in the N-terminal region which in other eukaryotic pld's is thought to interact with phosphoinositides in the membrane and to be involved with cell signalling (12,28).
When fungal pld's were subjected to phylogenetic analysis, Afpld 3 and its orthologues grouped separate -ly from Afpld 1 and Afpld 2 and their orthologues which themselves formed two sub-groups. Thus Afpld1 and Afpld 2may have arisen through a gene duplication event. Such gene duplications are commonplace in eukaryotes and are thought to play a pivotal role in the evolution of gene families (29,30).
Of the eukaryotic genes analysed, only A. fumigatus pld2 appeared to encode a signal peptide. Some bacterial PLD's are known to be secreted and play a role in pathogenesis. For example, Neisseria gonorrhoeae, a strict human pathogen, secretes a pld that is involved in membrane ruffling and is required for adhesion and cellular invasion (31). Similarly, extracellular pld's have also been shown to be involved in the virulence of Yersinia pestis and Corynebacterium pseudotuberculosis (32-34). Phosphatidic acid release through the action of secreted PLD in the lung, may account for some of the inflammatory responses observed following infection (4,35,36) as phosphatidic acid is known to act as a pro-inflammatory molecule stimulating the release of inflammatory cytokines (37-39).
In a study Li et al. (2012) showed PLD enzymes facilitate the invasion of Aspergills fumigatus to the lung tissue. They showed that the disruption of pld genes encoding PLD in A. fumigatus significantly decrease intracellular and extracellular phospholipase D activity (40). However, although indirect evidence of extracellular PLD activity in A. fumigatus was demonstrated, conclusive proof by partially sequencing the isolated protein will be needed and its significance in pathogenicity will have to be assessed by constructing a knockout strain and testing its virulence in a mouse model.